The Spliceosomal Phosphopeptide P140 Controls the Lupus Disease by Interacting with the HSC70 Protein and via a Mechanism Mediated by γδ T Cells
Figure 3
Ex vivo effect of P140 peptide on MRL/lpr PBL apoptosis.
(A) Induction of MRL/lpr and CBA/J PBLs apoptosis as measured by flow cytometry after annexin V- allophycocyanin labeling. The effect of increasing concentrations of the three peptides P140, 131–151 and ScP140 was assayed 20h after peptide incubation with cells. The results are expressed as the mean percentage (±SD) of specific apoptosis measured in three independent experiments. The decreased effect visualized when using 80 µM P140 probably results from peptide aggregation. (B) The same experiment was performed in measuring apoptosis of MRL/lpr PBLs by the decrease of mitochondrial Δψm, as measured by a reduction of DIOC6 dye uptake, in the presence of increasing concentrations of P140 peptide. (C) Induction of CD4+ and CD8+ T cell apoptosis by P140 peptide and effect of granzyme-B, perforin and caspase inhibitors. Twenty hours after P140 incubation, cell death was determined by flow cytometry using an annexin V-allophycocyanin labeling (see also insert c1). Granzyme-B inhibitor I (20 µMZ-AAD-CMK; see also insert c2), perforin inhibitor (5 nM concanamycin A) and caspase inhibitors (20 µM Z-VAD-FMK) were added 30 min post-P140 incubation to analyze the involvement of these pathways. The results are expressed as the mean percentage (±SD) of specific apoptosis measured in three independent experiments. P values were (1) <0.0001; (2) 0.0002; (3) non significant (ns); (4) ns; (5) 0.0003; (6) 0.0090. (D) Effect of P140 and 131–151 peptides on the viability of CD138+B220− plasmocytes as measured by flow cytometry 20h after MRL/lpr PBLs treatment. Plots are representative of three independent experiments. (E) Viability of murine lymphoma T cells T29 studied by measuring mitochondrial reduction of 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium dye 24h post-treatment with increasing concentrations of P140 peptide and two unrelated peptides X and Y used as positive controls. (F) Effect of P140 and 131–151 peptides on the B220 marker expression by CD4+ and CD8+ T cells. Expression of B220 was analyzed on these cells 20h after adding P140 or 131–151 peptides. The results are expressed as the mean percentage of cell subpopulations measured in three independent experiments. P values comparing CD4+B220− and CD8+B220− cells without or after P140 treatment were 0.059 and 0.0432, respectively. P values comparing CD4+B220+ and CD8+B220+ cells without or after P140 treatments were 0.0364 and 0.1707, respectively. The other comparisons were statistically non-significant.